1. Development of Mega-2, a working standard for factor VIII activity. Supplies of Mega-1, the U.S. working standard for coagulation factor VIII are low. The Center for Biologics Evaluation and Research (CBER), FDA has, therefore, initiated efforts to replace the Mega-1 standard with Mega-2. Mega-2 is a plasma-derived factor VIII concentrate. It was selected based on the integrity of the factor VIII molecule, linearity of the dose-response over a wide range of concentrations, minimal inter- and intra-assay variability, stability of factor VIII potency over accelerated and long term storage, and consistent results between different test methodologies (e.g. one-stage APTT assay versus chromogenic substrate assay). The final fill of Mega-2 was performed in May 2000, and 100,000 vials will be available for distribution. At present, CBER/FDA is collaborating with the European Directorate for the Quality of Medicines and the National Institute for Biological Standards and Control (NIBSC) in organizing the final phase of the potency calibration for Mega-2. The standard is anticipated to be ready for distribution in year 2001. 2. Development of the first international standard for von Willebrand factor (vWF) concentrates. The development of the first international standard for vWF concentrates is a joint effort among CBER/FDA, NIBSC, and the Science and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis. The initial characterization of five vWF concentrates was performed at three sites (CBER/FDA, NIBSC, and Laboratoire Francais du Fractionnement et des Biotechnologies). The results of the study were reported to the vWF subcommittee at the SSC annual meeting on June 16, 2000 in Maastricht, The Netherlands. The SSC has accepted the selection of two candidates for final phase of production and calibration. The two candidates were selected based on parallelism of dose-response curves, stability of preparations over accelerated and long term storage (i.e. stability of vWF multimers, antigen, and potency), similar results between different test methodologies (e.g. Ristocetin Cofactor Activity assay versus Collagen Binding assay), the ratio between activity and antigen, and the integrity of vWF multimers. A panel of 25-30 laboratories will be invited for the calibration studies. The final report of the study will be presented to the World Health Organization Expert Committee on Biological Standardization in October 2001. 3. Development of an International Thrombin Standard with an Unified Unit. The stock of the national thrombin standard, Lot J is very low and it needs to be replaced. The difference between current U.S. thrombin unit and international thrombin unit has been very confused and inconvenient for industry and scientists in academic environments (US ("NIH") UNIT = 1.152 X INTERNATIONAL UNIT). Initial discussion on the development of an international thrombin standard with an unified unit has been started among WHO, NIBSC and CBER. Drs. Ana Padilla from WHO and Elaine Gray from NIBSC have expressed great interest in collaboration with CBER/FDA in the development of a new international thrombin standard. 4. The Correlation of Closure Time and vWF Activities of Factor VIII/vWF Concentrates. SUMMARY OF WORK: To explore new test methodology, we investigated the PFA-100 system as a device for detection of vWF activity in factor VIII concentrates. Results: Seven factor VIII concentrates from 7 different manufacturers, and plasma cryoprecipitate were used in this study. VWF activities were tested by ristocetin cofactor (RCof) assay, for collagen binding, and for vWF antigen by ELISA. VWF multimers were detected by the electrophoresis of vWF on high resolution agarose gels (2%), followed by Western-blotting. To determine the relationship of closure time and vWF activity of factor VIII concentrates, a mixture of type III vWD blood (no detectable level of vWF) and factor VIII concentrate was incubated at room temperature for 10 minutes prior to testing on the PFA-100. Each dilution was tested on the PFA machine in duplicate with the collagen/epinephrine and collagen/ADP cartridges. We found that vWF with high molecular weight multimers plays an essential role for platelet anchorage to collagen and platelet aggregation in the PFA-100 system. Factor VIII concentrates with only low molecular weight multimers of vWF did not support the occlusion. There was no good correlation between the closure time and vWF:RCof activity of factor VIII concentrates. One unit of vWF:RCof activity did not always give identical closure time for the different factor VIII concentrates, suggesting that the shear stress assay measures the property of vWF which was not assessed by the RCof test. The relationship of the closure time and clinical outcome is under investigation. 5. Standardization of Recombinant Factor VIII. SUMMARY OF WORK: Several studies have shown that recombinant FVIII products often have discrepant potencies when different assay methods are applied. In the case of one recombinant product, use of the chromogenic assay, mandated in Europe, would mean that 30-50% less FVIII would be delivered to patients on a molar basis than if one applied the standard one-stage clotting assay used in the United States. It is important for us to discern the mechanism responsible for such discrepancies, to achieve standardization of potency measurements for recombinant FVIII. Results: Thrombin catalyzes the activation of FVIII by cleaving several peptide bonds located on the heavy and light chains of the FVIII molecule. To evaluate the effect of the acidic regions of FVIII on thrombin-catalyzed activation, we first asked whether there is a complementary binding site(s) on thrombin in addition to the catalytic site for FVIII. &#61537;-Thrombin, blocked at its active site by D-Phe-Pro-Arg Chloromethyl Ketone (PPACK), inhibited thrombin activation of FVIII. This suggests that a site(s) on thrombin, other than the active site, is needed for FVIII activation. Thrombin-catalyzed cleavages of both the heavy and light chains of FVIII were inhibited by a sulfated polypeptide segment of the FVIII heavy chain, residues 714-740 (F8II). F8II did not inhibit either the amidolytic or the clotting activity of thrombin. This further supports the involvement of sites on thrombin, other than the active site, in FVIII activation. To investigate the potential importance of the fibrinogen-recognition exosite of thrombin on FVIII cleavage, the C-terminal dodecapeptide of hirudin named Hirugen, which binds to the exosite, was tested as an inhibitor of thrombin proteolysis of FVIII. The peptide only partially inhibited the digestion of FVIII, mainly on the light chain, by &#61537;-thrombin. Furthermore, &#61543;-thrombin, which lacks the fibrinogen-recognition exosite, cleaved both the heavy and light chains of FVIII, at positions similar to those cleaved by &#61537;-thrombin. We conclude that a novel recognition site on thrombin, in addition of the fibrinogen-recognition exosite and the active site of thrombin, is involved in activation of FVIII. An acidic region (residues 714-740) of FVIII binds to one of the sites on thrombin involved in the rate-limiting step of the reaction.